Many of the products of biotechnology today are proteins and these proteins must be prepared in large volumes in purified form. The degree of purity required for proteins and other biomolecules for medical use is set by the national regulatory authorities, such as the US Food and Drug Administration (FDA). In addition to purity, the product must retain its biological activity, as the authorities will not certify a production procedure that results in variably active material. Thus, the process must produce the same amount and quality every time. To purify biomolecules, their inherent similarities and differences are often utilised. For example, protein similarity is used to purify them away from the other non-protein contaminants; while differences are used to purify one protein from another. Biomolecules such as proteins vary from each other in size, shape, charge, hydrophobicity, solubility, and biological activity.
One frequently used such method is chromatography, wherein two mutually immiscible phases are brought into contact. More specifically, the biomolecule is introduced into a mobile phase, which is contacted with a stationary phase. The biomolecule will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the components in the sample.
However, sometimes one technique for purifying a biomolecule is not enough. Two or more chromatography steps are often combined in series; and chromatography is advantageously combined with other techniques.
Precipitation is widely used for product recovery of biomolecules especially proteins. The most common type of precipitation of proteins is salt induced precipitation. Protein solubility depends on several factors. It is observed that at low concentration of the salt, solubility of the proteins usually increases slightly. This is termed “salting in”. But at high concentrations of salt, the solubility of the proteins drops sharply. This is termed “salting out” and the proteins precipitate out. A second method is the addition of an organic solvent. If there is a medium decrease in the dielectric constant with the addition of an organic solvent, the solubility should decrease also resulting in precipitation. A third method is precipitation by changing the pH of the protein solution. This effect is due to the different functional groups on a protein. There will be some pH, known as the isoelectric point where the net charge on the protein is zero. This is different for different proteins.
A specific example of protein precipitation is found in the purification of immunoglobulins, wherein traditional methods are often based on selective reversible precipitation of the protein fraction comprising the immunoglobulins while leaving other groups of proteins in solution. Typical precipitation agents are ethanol, polyethylene glycol, lyotropic i.e. anti-chaotropic salts such as ammonium sulphate and potassium phosphate, and caprylic acid. However, these precipitation methods are time-consuming and laborious. Furthermore, the addition of the precipitating agent to the raw material could make it difficult to use the supernatant for other purposes and creates a disposal problem, which is particularly relevant when speaking of large-scale purification of immunoglobulins.
U.S. Pat. No. 5,093,254 (Giuliano et al) relates to aqueous two-phase protein extraction employing polyvinylpyrrolidone (PVP) as the upper phase and maltodextrin as the lower phase. Thus, this is a two-polymer system, which is provided by mixing two aqueous solutions of PVP and maltodextrin at a temperature of 0-8° C. by vigorous mixing followed by centrifugation. The protein to be separated is then added to the two-phase PVP/maltodextrin system to which a dye has been added. The dye may be any amino derivative of triazine dyes, such as Cibacron Blue FGF, Procion Turquoise H-A and Procion Green HE-4BDA. After protein addition, the system is centrifuged to attain phase separation, resulting in the dye strongly partitioned to the upper PVP-containing phase.
The system should be operated at a temperature of 2-6° C. As the dye acts as an affinity ligand to the protein, it is in fact the dye-protein complex which is partitioned to the PVP-containing phase. Consequently, the protein can be extracted by separating the upper phase and elution of protein from dye e.g. by salt addition or pH increase. Thus, the '254 system requires centrifugation, which may affect the structure of more sensitive proteins. In addition, even though the '254 patent argues that their method is a low-cost system, the amount of preparation including mixing the twp polymers and preparing the dye still makes the use relatively time-consuming. In addition, the step of eluting proteins bound to the dye will require additional resources and time. Finally, another disadvantage of this system is the low operating temperature, which will require further demands on the equipment used.
WO 97/05480 (Massachusetts Institute of Technology) relates to a method, device and diagnostic kit for separating and/or concentrating an analyte from a mixture containing one or more contaminants according to size under a two-phase aqueous micellar system. In brief, the method disclosed includes providing at least one surfactant capable of forming a two-phase aqueous micellar system; forming a two phase aqueous micellar system; and permitting the analyte and the contaminant to partition unevenly between the two phases. The surfactant may be non-ionic, such as alkyl poly(ethylene oxide); zwitterionic (dipolar), such as dioctanoyl phosphatidylcholine; or ionic. The principle of excluded-volume interactions is utilised in WO 97/05480, meaning that conditions are selected which drives the majority of the larger reagent of the mixture into the aqueous domain of the micelle-poor phase while smaller reagents are driven into the aqueous domain of the micelle-rich phase. The disclosed method can be used for removing viruses from proteins following fermentation processes, as well as for concentrating viruses for vaccine manufacture or gene therapy.
U.S. Pat. No. 5,907,035 (Baxter Biotech Technology Sarl) relates to methods of purifying proteins having surface active, electron-rich amino acids, particularly histidine, from crude or partially purified protein solutions using an aqueous two phase system. The methods involve the use of polyethylene glycol (PEG), or similar inert hydrophobic molecules, conjugated to a metal chelator such as IDA, which is charged with a divalent metal ligand such as copper. The PEG-chelator-metal complex may be added directly to a crude protein solution containing the target protein. Salts and PEG may then be added and the solution is allowed to form a two-phase system. The target protein is recovered either from the salt or the polymer phase. However, the addition of large quantities of salt is usually a disadvantage, as firstly, salts are well known to denature proteins, and secondly, because a subsequent step for the removal thereof will be required.
WO 00/58342 (Valtion Teknillinen Tutkimuskeskus) relates to isolation and purification of proteins in aqueous two-phase systems (ATPS). Specifically, a process for partitioning proteins is provided by fusing said proteins to targeting proteins which have the ability to carry said protein into one of the phases. A stated advantage of the system is that it is inexpensive as a first or only step, which renders it suitable for the purification of proteins of relatively low market value such as enzymes.
Johansson et al (Hans-Olof Johansson et al: Thermoseparating Water/Polymer System: A Novel One-Polymer Aqueous Two-Phase System for Protein Purification, 1999 John Wiley & Sons) discloses an aqueous two-phase system which uses a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups at both ends (HM-EOPO). This polymer thermoseparates in water, forming an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO when separated at 17-30° C. The partitioning of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in the two-phase system was studied, and the amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO phase. The partitioning of hydrophobic proteins can be directed with addition of salt. The possibility of direct protein partitioning between water and copolymer phases shows that this system would be useful for protein separations.
U.S. Pat. No. 6,641,735 (Japan Chemical Innovation Institute) relates to a method for separating a target substance, for example, metal ion, drug or biological component using responsive polymers. According to the method, the surface of a packing undergoes a chemical or physical environmental change under a physical stimulus so that the interaction of a substance interacting with the target substance is reversibly changed in an aqueous solution, thus effecting separation.
However, as the biotechnology fields grows rapidly, and novel biomolecules are frequently presented, there is still a need in this field of alternative and advantageously improved methods for separation, either for combination with the prior art methods in a multi-step protocol of for use separately as single step protocols.